Michael-Christopher Keogh

A Snf2 Family ATPase Complex Required for Recruitment of the Histone H2A Variant Htz1

Deletions of three yeast genes, SET2, CDC73, and DST1, involved in transcriptional elongation and/or chromatin metabolism were used in conjunction with genetic array technology to screen approximately 4700 yeast deletions and identify double deletion mutants that produce synthetic growth defects. Of the five deletions interacting genetically with all three starting mutations, one encoded the histone H2A variant Htz1 and three encoded components of a novel 13 protein complex, SWR-C, containing the Snf2 family ATPase, Swr1. The SWR-C also copurified with Htz1 and Bdf1, a TFIID-interacting protein that recognizes acetylated histone tails. Deletions of the genes encoding Htz1 and seven nonessential SWR-C components caused a similar spectrum of synthetic growth defects when combined with deletions of 384 genes involved in transcription, suggesting that Htz1 and SWR-C belong to the same pathway. We show that recruitment of Htz1 to chromatin requires the SWR-C. Moreover, like Htz1 and Bdf1, the SWR-C promotes gene expression near silent heterochromatin.

Functional Organization of the S. cerevisiae Phosphorylation Network

Reversible protein phosphorylation is a signaling mechanism involved in all cellular processes. To create a systems view of the signaling apparatus in budding yeast, we generated an epistatic miniarray profile (E-MAP) comprised of 100,000 pairwise, quantitative genetic interactions, including virtually all protein and small-molecule kinases and phosphatases as well as key cellular regulators. Quantitative genetic interaction mapping reveals factors working in compensatory pathways (negative genetic interactions) or those operating in linear pathways (positive genetic interactions). We found an enrichment of positive genetic interactions between kinases, phosphatases, and their substrates. In addition, we assembled a higher-order map from sets of three genes that display strong interactions with one another: triplets enriched for functional connectivity. The resulting network view provides insights into signaling pathway regulation and reveals a link between the cell-cycle kinase, Cak1, the Fus3 MAP kinase, and a pathway that regulates chromatin integrity during transcription by RNA polymerase II.

Chaperone Control of the Activity and Specificity of the Histone H3 Acetyltransferase Rtt109

ABSTRACT Acetylation of Saccharomyces cerevisiae histone H3 on K56 by the histone acetyltransferase (HAT) Rtt109 is important for repairing replication-associated lesions. Rtt109 purifies from yeast in complex with the histone chaperone Vps75, which stabilizes the HAT in vivo. A whole-genome screen to identify genes whose deletions have synthetic genetic interactions with rtt109Δ suggests Rtt109 has functions in addition to DNA repair. We show that in addition to its known H3-K56 acetylation activity, Rtt109 is also an H3-K9 HAT, and we show that Rtt109 and Gcn5 are the only H3-K9 HATs in vivo. Rtt109s H3-K9 acetylation activity in vitro is enhanced strongly by Vps75. Another histone chaperone, Asf1, and Vps75 are both required for acetylation of lysine 9 on H3 (H3-K9ac) in vivo by Rtt109, whereas H3-K56ac in vivo requires only Asf1. Asf1 also physically interacts with the nuclear Hat1/Hat2/Hif1 complex that acetylates H4-K5 and H4-K12. We suggest Asf1 is capable of assembling into chromatin H3-H4 dimers diacetylated on both H4-K5/12 and H3-K9/56.

Bur1 kinase is required for efficient transcription elongation by RNA polymerase II

ABSTRACT The Saccharomyces cerevisiae cyclin-dependent kinase (CDK) Bur1 (Sgv1) may be homologous to mammalian Cdk9, which functions in transcriptional elongation. Although Bur1 can phosphorylate the Rpb1 carboxy-terminal domain (CTD) kinase in vitro, it has no strong specificity within the consensus heptapeptide YSPTSPS for Ser2 or Ser5. BUR1 mutants are sensitive to the drugs 6-azauracil and mycophenolic acid and interact genetically with the elongation factors Ctk1 and Spt5. Chromatin immunoprecipitation experiments show that Bur1 and its cyclin partner Bur2 are recruited to transcription elongation complexes, cross-linking to actively transcribing genes. Interestingly, Bur1 shows reduced cross-linking to transcribed regions downstream of polyadenylation sites. In addition, bur1 mutant strains have a reduced cross-linking ratio of RNA polymerase II at the 3′ end of genes relative to promoter regions. Phosphorylation of CTD serines 2 and 5 appears normal in mutant cells, suggesting that Bur1 is not a significant source of cotranscriptional Rpb1 phosphorylation. These results show that Bur1 functions in transcription elongation but may phosphorylate a substrate other than the CTD.

An acetylated form of histone H2A.Z regulates chromosome architecture in Schizosaccharomyces pombe

Histone variant H2A.Z has a conserved role in genome stability, although it remains unclear how this is mediated. Here we demonstrate that the fission yeast Swr1 ATPase inserts H2A.Z (Pht1) into chromatin and Kat5 acetyltransferase (Mst1) acetylates it. Deletion or an unacetylatable mutation of Pht1 leads to genome instability, primarily caused by chromosome entanglement and breakage at anaphase. This leads to the loss of telomere-proximal markers, though telomere protection and repeat length are unaffected by the absence of Pht1. Strikingly, the chromosome entanglement in pht1Δ anaphase cells can be rescued by forcing chromosome condensation before anaphase onset. We show that the condensin complex, required for the maintenance of anaphase chromosome condensation, prematurely dissociates from chromatin in the absence of Pht1. This and other findings suggest an important role for H2A.Z in the architecture of anaphase chromosomes.

Kin28 Is Found within TFIIH and a Kin28-Ccl1-Tfb3 Trimer Complex with Differential Sensitivities to T-Loop Phosphorylation

ABSTRACT Basal transcription factor TFIIH phosphorylates the RNA polymerase II (RNApII) carboxy-terminal domain (CTD) within the transcription initiation complex. The catalytic kinase subunit of TFIIH is a member of the cyclin-dependent kinase (Cdk) family, designated Kin28 in Saccharomyces cerevisiae and Cdk7 in higher eukaryotes. Together with TFIIH subunits cyclin H and Mat1, Cdk7 kinase is also found in a trimer complex known as Cdk activating kinase (CAK). A yeast trimer complex has not previously been identified, although a Kin28-Ccl1 dimer called TFIIK has been isolated as a breakdown product of TFIIH. Here we show that a trimeric complex of Kin28-Ccl1-Tfb3 exists in yeast extracts. Several Kin28 point mutants that are defective in CTD phosphorylation were created. Consistent with earlier studies, these mutants have no transcriptional defect in vitro. Like other Cdks, Kin28 is activated by phosphorylation on T162 of the T loop. Kin28 T162 mutants have no growth defects alone but do demonstrate synthetic phenotypes when combined with mutant versions of the cyclin partner, Ccl1. Surprisingly, these phosphorylation site mutants appear to destabilize the association of the cyclin subunit within the context of TFIIH but not within the trimer complex.

Using Chromatin Immunoprecipitation to Map Cotranscriptional mRNA Processing in Saccharomyces cerevisiae

The chromatin immunoprecipitation (ChIP) technique has been used to determine where and under what conditions DNA binding proteins associate with specific DNA sequences. Proteins are crosslinked in vivo with formaldehyde, and chromatin is then isolated and sheared. The protein of interest is then immunoprecipitated and the associated DNA sequences identified via PCR. Although this technique was originally designed to assay DNA binding proteins, it can also be used to monitor mRNA processing factors associated with transcription complexes.

Ctk1 promotes dissociation of basal transcription factors from elongating RNA polymerase II

As RNA polymerase II (RNApII) transitions from initiation to elongation, Mediator and the basal transcription factors TFIID, TFIIA, TFIIH, and TFIIE remain at the promoter as part of a scaffold complex, whereas TFIIB and TFIIF dissociate. The yeast Ctk1 kinase associates with elongation complexes and phosphorylates serine 2 in the YSPTSPS repeats of the Rpb1 C‐terminal domain, a modification that couples transcription to mRNA 3′‐end processing. The higher eukaryotic kinase Cdk9 not only performs a similar function, but also functions at the 5′‐end of genes in the transition from initiation to elongation. In strains lacking Ctk1, many basal transcription factors cross‐link throughout transcribed regions, apparently remaining associated with RNApII until it terminates. Consistent with this observation, preinitiation complexes formed on immobilized templates with transcription extracts lacking Ctk1 leave lower levels of the scaffold complex behind after escape. Taken together, these results suggest that Ctk1 is necessary for the release of RNApII from basal transcription factors. Interestingly, this function of Ctk1 is independent of its kinase activity, suggesting a structural function of the protein.

The Replication-independent Histone H3-H4 Chaperones HIR, ASF1, and RTT106 Co-operate to Maintain Promoter Fidelity

Background: Transcription is disruptive to chromatin structure and can expose cryptic promoters. Results: We identify those factors that might regulate cryptic transcription from within inactive and transcribed locations. Conclusion: Nucleosome shielding prevents cryptic transcription, and replication-independent histone replacement is co-operatively mediated by three H3-H4 chaperones. Significance: Understanding how cryptic transcription is regulated and lost histones replaced is of fundamental importance. RNA polymerase II initiates from low complexity sequences so cells must reliably distinguish “real” from “cryptic” promoters and maintain fidelity to the former. Further, this must be performed under a range of conditions, including those found within inactive and highly transcribed regions. Here, we used genome-scale screening to identify those factors that regulate the use of a specific cryptic promoter and how this is influenced by the degree of transcription over the element. We show that promoter fidelity is most reliant on histone gene transactivators (Spt10, Spt21) and H3-H4 chaperones (Asf1, HIR complex) from the replication-independent deposition pathway. Mutations of Rtt106 that abrogate its interactions with H3-H4 or dsDNA permit extensive cryptic transcription comparable with replication-independent deposition factor deletions. We propose that nucleosome shielding is the primary means to maintain promoter fidelity, and histone replacement is most efficiently mediated in yeast cells by a HIR/Asf1/H3-H4/Rtt106 pathway.

Corrigendum: A phosphatase complex that dephosphorylates |[gamma]|H2AX regulates DNA damage checkpoint recovery

This corrects the article DOI: 10.1038/nature04384